Journal: The Journal of Biological Chemistry

Article Title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains *

doi: 10.1074/jbc.M115.680009

Figure Lengend Snippet: Release of mature growth factor from BMP9·prodomain complex. Release of mature BMP9 domain after binding of an anti-pro-domain antibody was confirmed by application of BMP9·pro-domain to the pro-domain ELISA using anti-pro-domain antibodies AF3879 as capture and BAF3879 as detection (A). B, supernatant from A and recombinant mature BMP9 standard were applied to the mature BMP9 ELISA using anti-mature BMP9 antibodies MAB3209 as a capture and BAF3209 as detection. The mature BMP9 ELISA detected both mature BMP9 standard and released mature BMP9 from pro-domain ELISA supernatant.

Article Snippet: The animal facility is AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International)-accredited. . Antibodies used included anti-mature BMP9 antibodies MAB3209 and its biotinylated conjugate BAF3209 and anti-pro-domain antibodies AF3879 and its biotinylated conjugate BAF3879 and were purchased from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains *

doi: 10.1074/jbc.M115.680009

Figure Lengend Snippet: Release of mature growth factor from BMP9·prodomain complex. Release of mature BMP9 domain after binding of an anti-pro-domain antibody was confirmed by application of BMP9·pro-domain to the pro-domain ELISA using anti-pro-domain antibodies AF3879 as capture and BAF3879 as detection (A). B, supernatant from A and recombinant mature BMP9 standard were applied to the mature BMP9 ELISA using anti-mature BMP9 antibodies MAB3209 as a capture and BAF3209 as detection. The mature BMP9 ELISA detected both mature BMP9 standard and released mature BMP9 from pro-domain ELISA supernatant.

Article Snippet: Antibodies used included anti-mature BMP9 antibodies MAB3209 and its biotinylated conjugate BAF3209 and anti-pro-domain antibodies AF3879 and its biotinylated conjugate BAF3879 and were purchased from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: Origin of BMP9 expression. Immunostaining for BMP9 in human (a–f) and mouse (g, inset corresponds to rabbit control immunoglobulins) liver sections. Human and mouse tissue sections were stained with anti-BMP9 antibodies from R&D Systems (a, b, e, f, g) or from Biogenesis (c, d). The specificity of the staining was checked by addition of an excess of recombinant human BMP9 (1 μg/mL) (b, d). Note the strong immunostaining in hepatocytes and biliary ducts. In e, enlargement of a blood sinusoid [arrowhead indicates absence of staining in liver endothelial cells and arrows indicate staining in hepatocytes (H)]. In f, enlargement of a biliary duct [arrowhead indicates intrahepatic biliary epithelial cells and arrows indicate staining in hepatocytes (H)]. Slides were counterstained with haematoxylin. Scale bars 100 μm

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Expressing, Immunostaining, Control, Staining, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: BMP9 circulates in plasma under a high molecular mass form. a BMP9 biosynthesis. BMP9 is synthesized as a precursor protein (Pre-pro-BMP9) composed of 429 amino acids (aa) that include a 22 aa signal peptide, a 297 aa prodomain (33 kDa) and a 110 aa mature protein (12.5 kDa). The pre-pro-BMP9 then homodimerizes (pro-BMP9) and is subsequently cleaved by serine endoproteases. This generates two active forms: the short mature form (25 kDa) and the complexed form (100 kDa) in which the prodomain remains associated with the mature form. b Proteins from human plasma were separated by gel filtration chromatography. In a parallel experiment, recombinant human BMP9 was passed onto the same column. BMP9 activity was then measured in the different fractions using the ALK1-BRE-luciferase assay as described in “Materials and methods”. The data from one representative experiment (out of 4) are represented. c and d Plasma was passed through an anti-human serum albumin column. The plasma (1), the eluate (2) and the flow-through (3) were then analyzed by 10% SDS-PAGE and stained by Coomassie blue (c) and their BMP9 activity using the ALK1-BRE-luciferase assay was also quantified as described in “Materials and methods” (d). Data are expressed as the mean luciferase value ± SD obtained from two independent experiments

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Clinical Proteomics, Synthesized, Filtration, Chromatography, Recombinant, Activity Assay, Luciferase, SDS Page, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: BMP9 circulates in plasma under both active and inactive high molecular mass forms. a Proteins from a pool of five human plasmas were separated through gel filtration chromatography. BMP9 levels were then measured in the different fractions by three different means: the ALK1-BRE-luciferase assay, the BMP9 ELISA and the pro-BMP9 ELISA as described in “Materials and methods”. The ALK1-BRE-luciferase assay data and the data obtained from the BMP9 ELISA are presented as pg/mL of BMP9. The data obtained with the pro-BMP9 ELISA are presented as optical densities (OD). The data obtained in one representative experiment out of 3 are presented. b, c Human fractions (30–42) were treated with or without furin and BMP9 levels were then measured in the different fractions with the ALK1-BRE-luciferase assay (fraction 34, corresponding to the peak of BMP9 activity, was also measured c in the absence (gray square) or presence of anti-BMP9 neutralizing antibodies (black square). The ALK1-BRE-luciferase assay data are presented as pg/mL of BMP9. The data obtained in one representative experiment out of 2 are presented. d Nine human plasma (0.3%) were treated with or without furin and BMP9 activation was then measured using the ALK1-BRE-luciferase assay and the BMP9 ELISA as described in “Materials and methods”. Data are expressed as furin-treated plasma over untreated plasma mean ± SEM from duplicate determinations. e Pie chart representing the percentage of each BMP9 complex circulating in human plasma, as calculated from (d) measured with the ALK1-BRE luciferase assay

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Clinical Proteomics, Filtration, Chromatography, Luciferase, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: Ontogeny of BMP9 circulating levels in mice. BMP9 levels were measured from pooled diluted plasma (0.5%) taken from mice at the indicated developmental stages using the ALK1-BRE-luciferase assay as described in “Materials and methods”. In order to check that the activity measured by the ALK1-BRE-luciferase assay was attributable to BMP9, the assay was performed (inset) in the absence (gray squares) or the presence of anti-BMP9 neutralizing antibodies (black squares). The results are presented as means ± SD from triplicate determinations (E embryonic day, P post-natal day)

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Clinical Proteomics, Luciferase, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: Rat aortic endothelial cells are physiologically Smad1/5/8 phosphorylated in response to circulating BMP9. a Immunostaining for phosphoSmad1/5/8 of rat aorta cross-sections. The aortas were fixed immediately after surgical removal from the killed animal and processed for phospho-Smad1/5/8 immunostaining. Note the nuclear staining of endothelial cells. b Quantification of the number of phosphoSmad1/5/8-positive nuclei. Rat aortic slices were either fixed immediately after sacrificing the animal or incubated ex vivo for 1 h with PBS (step 1) and then for another hour with rat serum in the absence or presence of either anti-BMP9 neutralizing antibodies or recombinant ALK1ecd (step 2). The rat aorta slices where then fixed and immunostained for phosphoSmad1/5/8. Results are presented as the percentage of phosphoSmad1/5/8 nuclei in endothelial cells per aortic ring. Data are expressed as the mean ± SD of values obtained in 3 independent experiments (***p < 0.001)

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Immunostaining, Staining, Incubation, Ex Vivo, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: BMP9 mRNA expression in the different cell subtypes of human liver

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

doi: 10.1007/s00018-011-0751-1

Figure Lengend Snippet: BMP9 mRNA expression in nine human tissues

Article Snippet: Neutralizing anti-BMP9 antibodies (1 μg/mL, AF3209; R&D Systems) were incubated with plasma or fractions when indicated.

Techniques: Expressing